Membrane-bound ribosomes from BacIllus stearothermophilus contain an extra protein (M-protein) not found in free ribosomes. In vitro binding assays will be employed to determine if the M-protein is an essential requirement for the attachment of ribosomes to the membrane. The possibility that the M-protein plays a role in the vectorial transport of puromycin-released polypeptides across the membrane will also be investigated. Sepharose-immobilized trypsin will be used as a surface probe to detect both the ribosomal and membrane proteins located at the ribosome-interface. The hypothesis that an extracellular enzyme, alpha-amylase, is preferentially synthesized on membrane-bound polysomes will be tested. Nascent polypeptide chains synthesized in vitro on these polysomes as well as on polysomes not attached to membranes will be assayed for amylase-like protein both immunologically and chemically. If such site-specific synthesis can be demonstrated, membrane-bound and free ribosomes will be examined for the presence of messenger-discriminating species of initiation factor IF3. BIBLIOGRAPHIC REFERENCE: Miller, H. M., Friedman, S. M., Litman, D. J. and Cantor, C. R. Surface topography of the Bacillus stearothermophilus ribosome. Molec. Gen. Genet. 144: 273-280 (1976).